Metal-enhanced fluorescence from plastic substrates

ABSTRACT

The present invention relates to methods for functionally modifying a polymeric surface for subsequent deposition of metallic particles and/or films, wherein the polymeric surface is modified by increasing hydroxyl and/or amine functional groups thereby providing an activated polymeric surface for deposition of metallic particles to form a fluorescence sensing device. The device can be used for metal-enhanced fluorescence of fluorophores positioned above the metallic particles that can be readily applied to diagnostic or sensing applications of metal-enhanced fluorescence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending U.S. patentapplication Ser. No. 11/718,560, filed on Apr. 8, 2008, now U.S. Pat.No. ______, which is turn claims priority of International PatentApplication No. PCT/US2005/039498 is filed on Oct. 28, 2005, which inturn claims priority of U.S. Provisional Application No. 60/625,212filed on Nov. 5, 2004 and U.S. Provisional Patent Application No.60/630,992 filed on Nov. 24, 2004.

GOVERNMENT INTEREST

The invention disclosed herein was made with U.S. Government supportunder Grant No. GM070929-01 from the NIH. Accordingly, the U.S.Government has certain rights in this invention

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relate generally to a fluorescence sensing system,and more particularly, to a system including a polymeric surface havingmetallic particles deposited thereon and method of forming suchmetallizable polymeric surfaces.

2. Description of Related Art

Fluorescence systems have become a dominant technology in medicaltesting, drug discovery, biotechnology and cellular imaging. The use offluorescence technology has greatly enhanced the ability to detectspecific molecules leading to rapid advancements in diagnostics. Forexample, fluorescence detection is widely used in medical testing andDNA analysis because of the high degree of sensitivity obtained usingfluorescent techniques. Importantly a small numbers of molecules can bedetected using fluorescence technology. Typically, extrinsicfluorophores are added covalently or non-covalently to allow moleculesthat do not ordinarily fluoresce or do not fluoresce at useful levels tobe detected.

Detection of the molecule of interest is generally limited by theproperties of the fluorophore used. In some cases, labeling abiomolecule with an extrinsic fluorophore can alter the biologicalactivity of the biomolecule potentially creating experimental artifacts.Problems with current fluorescent techniques stem in part from the lowfluorescent intensities of commonly used fluorophores. Additionally,background fluorescence can be significant when using low wavelengthexcitation radiation required by some fluorophores or when largequantities of fluorophores are required.

At present, the use of noble metals, particles, and surfaces forapplications in sensing, is biotechnology, and nanotechnology has drawnconsiderable attention. For example, U.S. application Ser. No.10/073,625, discloses compositions and methods for increasingfluorescence intensity of molecules by adding either intrinsic orextrinsic fluorophores, and positioning same at a specific distance froma metal particle. Specifically, metal particles, deposited on glass orquartz type material, and biomolecules with a fluorophore are positionedat a distance from the metal particles. This positioning of thefluorophore at a specific distance from the metal particle can alter orincrease the intrinsic emission of electromagnetic radiation from thebiomolecule in response to an amount of exciting electromagneticradiation.

Favorable effects of silver particles on fluorophores include increasedquantum yields, decreased lifetimes, and increased photostability offluorophores commonly used in biological research. These effects ofconducting metallic particles on fluorescence have been the subject ofnumerous theoretical studies related to surface-enhanced Ramanscattering and the application of these considerations to molecularfluorescence. There is now interest in using the remarkable propertiesof metallic islands, colloids or continuous surfaces.

Consequently, it is of interest to develop convenient methods forforming metallic particles and/or films on different surfaces. Theseapproaches include electroless deposition, electroplating on insulators,lithography, and the formation of colloids under constant reagent flow.Metallic particles can be assembled into films using electrophoresis,and gold particles have been used for the on-demand electrochemicalrelease of DNA. It is anticipated that many of these approaches willfind uses in medical diagnostics and lab-on-a-chip-type applications.

Heretofore, all of these findings have been based on metallic silverbeing deposited on glass or quartz type substrates with a subsequentspacer layer used to separate the fluorophore from the metal. Thus, itwould be of great value to devise methods for localized or continuoussilver deposition on other surfaces that are more flexible that glassplanar surfaces or glass-based substrates.

SUMMARY OF INVENTION

The present invention relates to methods for functionally modifying apolymeric surface for subsequent deposition of metallic particles and/orfilms, wherein a polymeric surface comprising low density of functionalgroups is modified by increasing the number of hydroxyl and/or aminefunctional groups relative to an unmodified surface thereby providing anactivated polymeric surface useful for metal-enhanced fluorescence offluorophores positioned above the metallic particles that can be readilyapplied to diagnostic or sensing devices for applications ofmetal-enhanced fluorescence (MEF).

In another aspect, the present invention relates to uses of suchpolymeric surfaces for enhancement of effect of fluorophores nearmetallic surfaces comprising at least silver particles. These effectsinclude increased quantum yields, decreased lifetimes, increasedphotostability, and increased energy transfer. These effects are due tointeractions of the excited-state fluorophores with the surface plasmonresonances on the metallic surfaces. These interactions of thefluorophore with the metal surface can result in increased rates ofexcitation, quenching, increased intensities, and/or increased quantumyield.

In yet another aspect, the present invention relates to a method fordepositing a noble metal on a polymeric surface, the method comprising:

-   -   a) providing a polymeric material with low density of hydroxyl        surface functionality;    -   b) modifying the polymeric material with a chemical or physical        etching agent, wherein the modified polymeric material comprises        an increased number of exposed hydroxyl groups or amine group on        the polymeric surface relative to an unmodified surface;    -   c) silylating the modified polymeric material to provide an        amino-terminated polymeric material; and    -   d) depositing a noble metal on the amino-terminated polymeric        material.

Subsequent to the deposition of the noble metal particles, a spacer canbe applied to the metal surface to provide a required distance from themetal surface to the fluorophore for metal enhanced fluorescence.

In yet another aspect, the present invention relates to a method forforming a patterned metallic surface on a polymeric substrate, themethod comprising:

-   -   (a) providing a polymeric substrate;    -   (b) etching the surface of the polymeric substrate with an        etching agent to increase hydroxyl groups on the surface of        polymer substrate relative to a non-etched polymeric surface    -   (c) contacting the polymeric substrate having increased hydroxyl        groups with a silylating agent to replace at least some of the        hydroxyl groups with amine groups to form an amino-activated        surface; and    -   (d) depositing noble metal particles on the amine activated        polymeric substrate.

A still further aspect of the present invention relates to afluorescence sensing device comprising:

-   -   a modified polymeric surface comprising amino-terminated        functional groups;    -   nanometer sized metallic particles deposited on the modified        polymeric surface, wherein the metallic particles can be any        geometric shape; and    -   a fluorophore compound communicatively linked to the metal        particles at a sufficient distance to enhance the fluorescence        of the fluorophore when exposed to electromagnetic radiation        from an electromagnetic source.

The present invention further comprises a detection device for detectingfluorescence emissions including, but not limited to visual inspection,digital (CCD) cameras, video cameras, photographic film, or the use ofcurrent instrumentation such as laser scanning devices, fluorometers,luminometers, photodiodes, quantum counters, plate readers,epifluorescence microscopes, scanning microscopes, confocal microscopes,capillary electrophoresis detectors, or other light detectors capable ofdetecting the presence, location, intensity, excitation and emissionspectra, fluorescence polarization, fluorescence lifetime, and otherphysical properties of the fluorescent signal.

A source of electromagnetic energy may include lasers emitting radiationfrom the UV to IR spectrum, masers, LEDs, incandescent lamps, etc, andwhich will be determined by the frequency or wavelength of energyrequired for excitation of the specific fluorophore.

A further aspect of the present invention, relates to a kit fordetecting a target molecule in a sample, the kit comprising

-   -   a container comprising a layer of immobilized metal particles        deposited on a polymeric substrate, wherein an immobilized probe        is connected to the metal particles and wherein the immobilized        probe has an affinity for the target molecule; and    -   a fluorophore having an affinity for the target molecule,        wherein the binding of the target molecule to both the        immobilized probe and fluorophore causes the fluorophore to be        positioned a sufficient distance from the immobilized metal        particles to enhance fluorescence emission,

Other aspects and advantages of the invention will be more fullyapparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows photographs of two (2) plastic films mounted on glassslides. Left—silver island films (SiFs) deposited on unmodified PC andRight—SiFs deposited on NaOH etched PC.

FIG. 2 shows absorption spectra of Silver Island Films, SiFs, grown onboth modified PC and glass.

FIG. 3 shows emission spectra of FITC-HSA monolayers on modified PC withand without SiFs and on virgin PC (unmodified). The transmission spectraof the 500 nm cut-off filter is also shown.

FIG. 4 shows photographs: SiF coated modified plastic, Top; the emissionof fluorescein labeled human serum albumin (FITC-HAS) on the unsilveredmodified PC, Middle; and on the silvered and modified PC, Bottom.

FIG. 5 shows the intensity decays of FITC-HSA on silvered and unsilveredmodified PC: The instrumental response Function, IRF, is also shown.

FIG. 6 shows emission intensity Vs time of FITC-HSA on both silvered andunsilvered modified PC with constant 470 nm excitation, Top; and withthe laser power adjusted to give the same initial steady statefluorescence intensity, Bottom.

DETAILED DESCRIPTION OF THE INVENTION

In the past several years a number of different metal-fluorophorecombinations and geometries [1-5] have been developed, which ultimatelyyielded significantly brighter and more photostable fluorophores. Theseadvances were not due to chemical structure modifications, but are dueto the control of the intrinsic fluorophore radiative decay rate.Specifically, this new technology has been named this metal-enhancedfluorescence [1,5] and radiative decay Engineering (RDE) [2,3].Primarily with the use of MEF, silver nanostructures deposited ontoclean glass microscope slides or quartz plates have been used. This hasbeen because the chemistries of the surface of glass are wellestablished and therefore the covalent immobilization of silvernanostructures onto glass less arduous and indeed reproducibly reliable.

Polymer substrates are known to be very promising substrates for avariety of applications and industrial interest in utilizing plastics isprimarily driven by the fact that these materials are less expensive andeasier in mass production than silica based substrates [6]. There arealso a wide variety of materials to choose from with an even greaterarray of chemical and physical properties [6].

The present invention shows that polymeric substrates can be used assubstrates for metal-enhanced fluorescence, which, given their cost, arelikely to be much better received by industry. While high surfacedensity of either hydroxyl or amino groups, which are readily used forsilver deposition, the present invention provides modification of apolymeric surface exhibiting low density of surface functionality. Thismodification allows for MEF to be introduced into already existingplastic based technologies, such as with plastic high-throughputscreening well plates and fluorescence based clinical assays. Thepractical approaches to polymer surface modification are coronadischarge treatment, plasma, surface graft, light and chemicalmodification [12].

The presence of a nearby metal film, island or particle can also alterthe emission properties of fluorophores. The most well known effect isthe quenching of fluorescence by a near-by metal. The emission offluorophores within 50 Å of a metal surface is almost completelyquenched. This effect is used in fluorescence microscopy with evanescentwave excitation. For a fluorophore located on a cell membrane and nearthe quartz-water interface the fluorescence emission is quenched,allowing selective observation of the emission from a fluorophore in thecytoplasmic region of the cell, which is more distant from thesolid-liquid interface. In addition to quenching, metal surfaces orparticles can cause significant increases in fluorescence. Remarkably,depending on the distance and geometry, metal surfaces or particles canresult in enhancement factors of up to 1000 for the fluorescenceemission [17-19]. Fluorophores near a metal film are not expected toemit isotropically, but rather the emission is directed into selecteddirections that are dependent on the sample configuration and the natureof the metallic surface. In addition to directionality, the decay timesof fluorophores are altered by the metal. In fact, the lifetimes offluorophores placed at fixed distances from a continuousmetallic-surface oscillate with distance [20].

The effects of metallic particles and surfaces on fluorophores are dueto at least three known mechanisms. One mechanism is energy transferquenching, k_(m), to the metals with a d⁻³ dependence. This quenchingcan be understood by damping of the dipole oscillations by the nearbymetal. A second mechanism is an increase in the emission intensity dueto the metal increasing the local incident field on the fluorophore,E_(m), with a maximum theoretical enhancement effect of about 140. Thiseffect has been observed for metal colloids and is appropriately calledthe “Lightning Rod effect.” This enhancement can be understood as due tothe metal particles on concentrating the local field and subsequentlyincreasing the rate of excitation. The third mechanism is that a nearbymetal can increase the intrinsic decay rate of the fluorophore, Γ_(m),that is, to modify the rate at which the fluorophore emits photons.These later two fluorophore-metal interactions offer remarkableopportunities for advanced fluorescence assay-technology.

The distance dependence of fluorescence enhancements and those ofquenching may be determined by standard methods disclosed herein.

In fluorescence, the spectral observables are governed by the magnitudeof Γ, the radiative rate, relative to the sum of the non-radiative decayrates, k_(nr) such as internal conversion and quenching. In the absenceof metallic particles or surfaces, the quantum yield, Q₀ andfluorescence lifetime τ₀ are given by:

$Q_{0} = \frac{\Gamma}{\Gamma + k_{nr}}$$\tau_{0} = \frac{1}{\Gamma + k_{nr}}$

Fluorophores with high radiative rates have high quantum yields andshort lifetimes Increasing the quantum yield requires decreasing thenon-radiative rates k_(nr), which is often only accomplished when usinglow solution temperatures or a fluorophore binding in a more rigidenvironment. The natural lifetime of a fluorophore, τ_(N), is theinverse of the radiative decay rate or the lifetime, which would beobserved if the quantum yield were unity. This value is determined bythe oscillator strength (extinction coefficient) of the electronictransition [21-24]. The extinction coefficients of chromophores are onlyvery slightly dependent on their environment. Hence, for almost allexamples currently employed in fluorescence spectroscopy, the radiativedecay rate is essentially constant.

The concept of modifying the radiative decay rate of fluorophores isunfamiliar to most spectroscopists. It is therefore intuitive toconsider the novel effects of fluorescence enhancement due to metalparticles, m, by assuming an additional radiative rate, Γ_(m), as shownin FIG. 3. In this case, the quantum yield and lifetime are given by:

$Q_{m} = \frac{\Gamma + \Gamma_{m}}{\Gamma + \Gamma_{m} + k_{nr}}$$\tau_{m} = \frac{1}{\Gamma + \Gamma_{m} + k_{nr}}$

These equations result in important predictions for a fluorophore near ametal surface. As Γ_(m) increases, the fluorescence quantum yieldincreases while the lifetime decreases, as shown in FIG. 4, which isconverse to the free space condition where both change in unison. Anability to modify and control the radiative decay rate (Γ+Γ_(m)) canhave profound implications for the use of fluorescence in basic researchand its applications.

The reduction in lifetime of a fluorophore near a metal is due to aninteraction between fluorophore and metal particle, which enhances theradiative decay rate (quantum yield increase) or depending on distance,d⁻³, causes quenching. A shorter excited-state lifetime also allows forless photochemical reactions which subsequently results in increasedfluorophore photostability.

Fluorophore photostability is a primary concern in many applications offluorescence. This is particularly true in recent trends in singlemolecule spectroscopy. A shorter lifetime also allows for a largerphoton flux. The maximum number of photons that are emitted by afluorophore each second is roughly limited by the lifetime of itsexcited state. For example, a 10 ns lifetime can yield about 10⁸ photonsper second per molecule, but in practice, only 10³ photons can readilybe observed. The small number of observed photons is typically due toboth photodestruction and isotropic emission. If the metal surfacedecreases the lifetime then one can obtain more photons per second permolecule by appropriately increasing the incident intensity. On theother hand, the metal enhanced fluorescence effects of the presentinvention enhances intensity while simultaneously shortening thelifetime. Decreases in the excitation intensity will still result inincreases in the emission intensity and therefore photostability.

The ability to increase the radiative decay rate suggests that anychromophore, even non-fluorescent species such as bilirubin, fullerenes,metal-ligand complexes or porphyrins could display usefully high quantumyields when appropriately placed near a metal surface.

The effects of metal surface-fluorophore interactions are highlydependent upon distance and the nature of the metal surface. Theemission enhancement is observed when fluorophore distances near 4-50 nmto the metal surfaces. At this scale, there are few phenomena thatprovide opportunities for extremely high levels of assay—sensing,manipulation, and control. In addition, devices at this scale may leadto dramatically enhanced performance, sensitivity, and reliability withdramatically decreased size, weight, and therefore cost, importantconsiderations for field-deployable bio-terrorism anthrax sensors.Slightly different effects can be expected for mirrors, sub-wavelengthor semi-transparent metal surfaces, silver island films or metalcolloids.

Before the present invention is disclosed and described, it is to beunderstood that this invention is not limited to the particular processsteps and materials disclosed herein as such process steps and materialsmay vary somewhat. It is also to be understood that the terminology usedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include pluralreferences unless the content clearly dictates otherwise.

The term “fluorophore” means any substance that emits electromagneticenergy such as light at a certain wavelength (emission wavelength) whenthe substance is illuminated by radiation of a different wavelength(excitation wavelength). Extrinsic fluorophores refer to fluorophoresbound to another substance. Intrinsic fluorophores refer to substancesthat are fluorophores themselves. Exemplary fluorophores include but arenot limited to those listed in the Molecular Probes Catalogue, which isincorporated by reference herein. Representative fluorophores includebut are not limited to Alexa Fluor® 350, Dansyl Chloride (DNS-Cl),5-(iodoacetamida)fluoroscein (5-IAF); fluoroscein 5-isothiocyanate(FITC), tetramethylrhodamine 5-(and 6-)isothiocyanate (TRITC),6-acryloyl-2-dimethylaminonaphthalene (acrylodan),7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium bromide,Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamineB sulfonyl chloride, Texas Red™, sulfonyl chloride, naphthalaminesulfonic acids including but not limited to1-anilinonaphthalene-8-sulfonic acid (ANS) and6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS), Anthroyl fatty acid,DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid,Fluorescein-phosphatidylethanolamine, Texasred-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,Fluorenyl-phosphotidylcholine, Merocyanine 540,1-(3-sulfonatopropyl)-4-[-.beta.-[2 [(di-n-butylamino)-6naphthyl]vinyl]pyridinium betaine (Naphtyl Styryl),3,3′dipropylthiadicarbocyanine (diS-C₃-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, rhodamine 800, IR-125,Thiazole Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine 1, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33342, TOTO, Acridine Orange,Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE),Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofluors,Coronene, and metal-ligand complexes.

Representative intrinsic fluorophores include but are not limited toorganic compounds having aromatic ring structures including but notlimited to NADH, FAD, tyrosine, tryptophan, purines, pyrirmidines,lipids, fatty acids, nucleic acids, nucleotides, nucleosides, aminoacids, proteins, peptides, DNA, RNA, sugars, and vitamins. Additionalsuitable fluorophores include enzyme-cofactors; lanthanide, greenfluorescent protein, yellow fluorescent protein, red fluorescentprotein, or mutants and derivates thereof.

In accordance with this invention, any flexible or rigid polymericsubstrate can be utilized in the process of this invention. Typical filmor relatively rigid substrates include polymeric compositions containingpolyamide, polycarbonate, polyester, polyetherimide, polyimide,polynitrocellulose, polyolefins such as polyethylene, polypropylene,poly(ethylenevinylacetate), poly-2-pentene, EPDM, polyionomers such asSurlyn®, polyphenylene oxide, polyphenylene sulfide, polysulfone,polystyrene, polyvinyl-vinylidine chloride or fluoride or the like.

Alternatively, rigid substrates may include any rigid substrates coatedwith a polymeric surface including, but not limited to, ceramics, glass,paper compositions or the like; or composite substrates such asepoxy-fiber glass, epoxy-paper laminate, paper-fiber glass laminate,urea formaldehyde-fiber glass laminate, phenolic-fiber glass laminate, apolymeric fluorocarbon-fiber glass laminate, or the like or with otherreinforcing components such as carbon fiber, synthetic polymer fiber,pigments or the like.

The preferred substrates include polycarbonates, polyesters,polyetherimides, polyimide, polyolefins or polysulfone. More preferably,the substrate is a polymeric material that upon treatment with anactivation agent increases hydroxyl groups on the surface of thepolymeric substrate. Most preferably, the polymeric substrate includespolyimides (Kapton®); polyesters (Mylar®); polycarbonates (Lexan®) andpolyetherimides (Ultem®) due to their physical and thermal stabilityover wide temperature ranges, chemical inertness and radiationresistance.

It may be necessary to pre-treat the polymeric substrate in order toremove any unwanted surface contamination before the functionalizationprocess. For example, the surface of polyethylene is typicallycontaminated with low molecular-weight, wax-like, incompletelypolymerized oligomers of ethylene, the monomer for polyethylene. Thesepoorly adherent fragments should be removed and can be easily andquickly degraded into volatile compounds and can be removed by shortplasma treatment thereby leaving the polymeric surface essentiallyintact and minimally etched if short treatment times are used. Forexample, argon may be used since the plasma treatment to removecontaminants and it time is relatively short thereby reducing anyunwanted chemistry.

A typical cleaning procedure for example polyethylene would be to treatwith Argon at a pressure of 0.01 to 0.4 Torr, with a power density ofabout 0.5 W/cm² at 13.56 MHz rf on parallel-plate electrodes. Oncecontaminants are removed, if necessary, a more stable polymer surface isexposed to an activating agent to provide an increased density ofhydroxyl or amine functional groups of the surface of the polymericsubstrate, relative to a polymeric surface without the activationtreatment. [16].

After cleaning or removal of contaminants, if necessary, of the surfaceof the polymeric substrate, etching is used to increase the density offunctional groups on the surface substrate. The polymeric substrate isetched to provide attraction sites for subsequent catalytic metaldeposition.

Etching involves solvating the polymeric substrate with a solvent tochemically modifying the surface substrate to provide attraction sitesfor catalytic metal deposition. For the present invention, a widevariety of etchants are satisfactory as long as selective solvation andchemical modification occurs. Typically, the first step in this processis to create hydroxyl groups (if they do not already exist on thesupport) or amino groups on the support. Surface activation comprisesformation of reactive hydrogen groups in a surface region of thesubstrate, wherein the reactive hydrogen groups are selected from one ormore members of the group comprising OH, OOH and COOH groups.

Some polymeric surfaces may be activated by a wet process, comprisinghydrolysis by a dilute aqueous base, such as NaOH, NH₄OH, LiOH, KOH orN(CH₃)₄OH. Preferably, the base hydrolysis of the polymeric surface isperformed in aqueous NaOH at a temperature ranging from about 15° C. toabout 45° C. for a time ranging from about 10 minutes to about 5 hours,and more preferably from about 1 minute to one hour depending on thepolymeric material.

In another embodiment, the surface activation step of the presentinvention may comprise formation of reactive hydrogen groups in asurface region of the polymeric surface by photo-oxidation, whichpreferably comprises irradiation of the surface of the substrate with adose of UV radiation, in the presence of oxygen.

UV radiation comprises radiation in the region of the electromagneticspectrum including wavelengths from about 100 to about 380 nm. Thepreferred wavelengths to which substrates are exposed in the activationstep is variable, and depends on the composition of the specificsubstrate. For example, polyimides or polycarbonates are preferablyirradiated with UV radiation having wavelengths from about 200 to about300 nm.

Preferably, the source of UV radiation is a low-pressure quartz-mercurylamp having an intensity of from about 1 to about 5 mW/cm². The durationof the UV exposure is preferably from about 1 minute to about 120minutes, more preferably from about 2 to about 20 minutes. The preferredUV dose is from about 0.7 J/cm² to about 5 J/cm², more preferably fromabout 2 to about 5 J/cm², depending on the substrate and the amount offunctionalization desired. These parameters are preferred for productionof reactive hydrogen groups. Irradiation for longer times and/or athigher intensities can result in a decrease in the amount of activehydrogen groups and an increase in the amount of other oxygen-containinggroups such as (ketone) carbonyl groups.

The activation of the polymeric surface substrate in the presence of UVradiation is believed to be a result of simultaneous excitement ofmolecules comprising the substrate and attack by molecular oxygen, aswell as ozone, atomic oxygen and singlet oxygen generated from molecularoxygen by UV radiation.

As a result of the photochemically-induced oxidative surfacemodification, oxygen-containing reactive hydrogen groups such as OH, OOHand COOH are formed on the surface of the substrate. Preferably, surfaceactivation of the substrate occurs to a depth of about 200 to about 1000nm below the surface of the substrate, producing a surface region of thesubstrate containing active hydrogen groups.

It is possible to monitor the progress of the surface activationreaction by measuring the water contact angle of the substrate surfaceat different times during the activation step because the water contactangle decreases due to the increased hydrophilicity of the polymersurfaces.

Although the surface activation substrates has been described above withreference to oxidative activation by molecular oxygen and UV and a wetprocess for polyimides, it is to be understood that other processes maybe used to activate the polymeric surface of a substrate. For example,dry processes such as oxidation in oxygen containing plasmas, oxygenion-beam modification, oxidation by fast atomic oxygen (FAO), and coronadischarge may be used to produce reactive hydrogen groups on the surfaceof a solid substrate.

Next the activated polymeric surface having an increased number ofhydroxyl groups is silylated by reacting at least some of the reactivehydrogen groups, formed in the upper region of the substrate by thesurface activation step, with a silylating agent, wherebysilicon-containing groups of the silylating agent become chemicallybonded to polymer molecules in the surface region of the substrate.(Notably, the term “silanization” is interchangeable with the term“silylation”).

The silylation step according to the present invention may preferably becarried out as a vapor phase or liquid phase reaction, preferably usinga silylating agent containing organosilicon groups and selected from thegroup comprising monofunctional and polyfunctional silylating agentsthat include amino groups.

Preferred monofunctional silylating agents include3-(aminopropyl)triethoxysilane (APS), dimethylsilyldimethyl amine(DMSDMA), 1,1,3,3-tetramethyl disilazane (TMDS), N,N-dimethylaminotrimethylsilane (TMSDMA), N,N-diethylaminotrimethylsilane (TMSDEA) andhexamethyldisilazane (HMDS). Preferred polyfunctional silylating agentsinclude Bis(dimethylamino)methylsilane,Bis-(dimethylamino)dimethylsilane and1,1,3,3,5,5-hexamethylcyclotrisilazane (HMCTS).

Gas phase silylation is preferably carried out in a vapor of silylatingagent, most preferably in a nitrogen carrier gas at elevatedtemperatures, preferably in the range of about 140 to 200° C.

Notably, in the process of the present invention, liquid-phasesilylation may also be used. The liquid phase silylation solution iscomprised of two and possibly three components: 1) the silylating agent,2) the transport solvent, and possibly, 3) a diffusion enhancer. Thesilylating agent is, as previously outlined, the chemical agent thatcarries the necessary silicon for binding to the activated polymericsurface. The transport solvent acts as the solvent for the silylatingagent, and should be relatively inert otherwise. The diffusion enhanceris a solvent that dissolves the surface of the substrate slightly,allowing the silylating agent to diffuse deeper and more rapidly belowthe surface of the substrate, preferably throughout the entire surfaceregion containing reactive hydrogen groups.

Preferred transport solvents are those that act as a solvent for thesilylating agent, and are inert toward the substrate, that is, they donot dissolve or swell the substrate. The most preferred solvents arehydrocarbons, such as ethanol and aromatic solvents such as xylene, andaliphatic solvents such as n-decane.

Preferably, the silylating agent diffuses into the substrate to reactwith active hydrogen atoms throughout the activated surface region ofthe substrate. The diffusion rate of the silylating agent may preferablybe increased by slight heating, up to about 60° C., and by the addition,to the silylation bath, of the diffusion enhancer. For polymers such asKapton, PEEK and PET a diffusion enhancer such as n-methylpyrrolidone(NMP) can be added. Silylation is generally carried out at 50-80° C.,the substrate being immersed in a solution of silylating agent for about3 minutes to 24 hours.

After silylation, the polymeric surface is ready for deposition ofmetallic particles. The metal particles used in the present inventioncan be spheroid, ellipsoid, triangular or of any other geometry andpreferably are deposited on the polymeric surface to form small islands.Metal particles, preferably noble metals, most preferably silver, may bechemically reduced on a surface.

The island particles may be prepared in clean beakers by reduction ofmetal ions using various reducing agents. [25]. For example, sodiumhydroxide is added to a rapidly stirred silver nitrate solution forminga brown precipitate. Ammonium hydroxide is added to re-dissolve theprecipitate. The solution is cooled and dried polymeric substrates areadded to the beaker, followed by glucose. After stirring for 2 minutes,the mixture is warmed to 30° C. After 10-15 minutes, the mixture turnsyellow-green and becomes cloudy. A thin film of silver particles hasformed on the polymeric surfaces and then the polymeric substrate isrinsed with pure water prior to use.

Alternative procedures for preparing metal particles are also available[26-30]. Silver is primarily used because of the familiar color from thelonger surface plasmon absorption of silver.

Determining the correct positioning of the fluorophore attached to atarget molecule relative to the metallic particle is essential formaximum fluorescence enhancement geometries (distance dependence). Thepresent inventors have previously conducted calculations for severalprobes. Similar calculations may also be done for many othercommercially available fluorophores. By controlling the fluorophoreenvironment, such as modifying the pH, the functional properties of themetallized polymeric surface/fluorophore sensor in terms of enhancedfluorescence and improved photostability may be determined. After eachenvironmental change, spectroscopic data may be acquired, analyzed andassessed in terms of the probe functionality in various nano-sites. Suchmeasurements will allow immediate comparison of the fluorophore and therelative distance that display substantial enhancement due to theappropriate proximity to the metal surface and those which are notaffected (i.e. too far from metal surface) and can be used forfluorescence sensors on the nanometer scale.

Once the appropriate distance is determine between the fluorophore andmetallic particle, the distance may be maintained by usingLangmuir-Blodgett films with fatty acid spacers. The fatty acids may befrom natural sources, including concentrated cuts or fractionations, orsynthetic alkyl carboxylic acids. The Langmuir-Blodgett techniqueprovides an accurate means of controlling film thickness and surfaceuniformity and allows an accurate control of the metal-fluorophoredistance.

Further, metal-fluorophore distances may be achieved by using polymerfilms. Examples of the polymer include, but not limited to, polyvinylalcohol (PVA). Absorbance measurements and ellipsometry may be used todetermine polymer film thickness. One type of polymer films may includespin coated polymer films.

The film spacer layer may be one or multiple layers formed from anoxide. The oxide layer may be formed from a deposition technique, suchas vapor deposition. Preferably, the oxide is a silicon oxide, morepreferably, SiO₂. The vapor deposition of SiO₂ is a well establishedtechnique for the controlled deposition of a variety of substrates.

Further, proteins or oligonucleotides may be bound to silver surfaces orparticles by using amino or sulfhydryl groups using methods known in theart. The length of the complimentary captured protein oroligonucleotide, within the enhancement region (40-500 Å), can also bedetermined by the metal enhanced fluorescence experiments with theLangmuir-Blodgett films and spin coated PVA, as discussed above.Fluorophore-metal distances that provide maximum fluorescenceenhancement are determined empirically and are thus used in determiningthe amino acid or DNA sequence length to use for capturing the sequencesso that fluorescence of same is optimally enhanced.

Detection devices applicable for detecting fluorescence emissionsinclude, but not limited to visual inspection, digital (CCD) cameras,video cameras, photographic film, or the use of current instrumentationsuch as laser scanning devices, fluorometers, luminometers, photodiodes,quantum counters, plate readers, epifluorescence microscopes, scanningmicroscopes, confocal microscopes, capillary electrophoresis detectors,or other light detector capable of detecting the presence, location,intensity, excitation and emission spectra, fluorescence polarization,fluorescence lifetime, and other physical properties of the fluorescentsignal.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES

A polycarbonate film (PC) was modified using chemical modification forsilver deposition and therefore metal-enhanced fluorescence. Basecatalyzed hydrolysis of the PC film readily created additional surfacefunctionality for silver island film deposition. Polycarbonate waschosen as the polymer of interest due to its widespread use inbiotechnology [6,12,13].

Metal-enhanced fluorescence is known to be a through space phenomenon,where the close proximity of fluorophores to silver nanostructuresresults in quenching of the emission [1, 2, 5]. The fluorophore waspositioned about 4 nm from the metallic surface using a labeled protein,namely FITC-HSA. The disclosed results clearly show that plastics canindeed be modified for silver deposition and notable enhancements influorescence emission can be achieved from the plastic substrates,similar to that observed from glass [5].

Materials and Methods

Polycarbonate (PC) films with ≈50 μm thickness were cut into 75×25 mmpieces and placed onto Fisher brand glass microscope slides in order toprovide support for the films. PC films were hydrolyzed in 2 M aqueousNaOH solution for 1 minute and rinsed with deionized water. PC filmswere then transferred onto new glass slides and finally dried under astream of cooled air. The hydrolyzed PC films were silanized with a 2%v/v solution of 3-(aminopropyl)triethoxysilane (APS) in denaturedethanol for 2 hours. The APS coated PC films were removed from thesolution and rinsed several times with ethanol and deionized water toremove the unbound APS. Silver Island Films (SiFs) were formed on halfof the silanized PC films (the non-silvered half is used as a control)similar to our previous procedure [3,5]. SiFs were also formed on virginPC films, i.e. unmodified films.

In previous reports of metal-enhanced fluorescence (MEF), silvered glassor quartz surfaces were coated with fluorophore labeled protein [5].This same experimental format has been adopted for two main reasons, thefirst, being that the protein coverage with Human Serum Albumin (HSA) isknown to bind to silvered surfaces and indeed forms a monolayer [4,5]and secondly, the dimensions of the protein is being such that theprotein allows for a mean ≈4 nm separation of the silver and thefluorophore, MEF being a through space phenomenon [1,2,5].

Binding the FITC-HSA to the PC films was accomplished by soaking in a 10uM FITC-HSA solution for 2 hours, followed by rinsing with water toremove the unbound material. PC films were then transferred onto newglass slides. Both the unsilvered and silvered PC films were coated withlabeled HSA, which is known to passively absorb to noble metal surfacesand form a ≈4 nm thick protein monolayer, allowing us to study thefluorescence spectral properties of noncovalent FITC-HSA complexes inthe absence and presence of SiFs. By equally coating a PC film withFITC-HSA determination of the enhancement factor (benefit) obtained fromusing the silver, i.e. Intensity on Silver/Intensity on PC film, giventhat both surfaces are known to have an equal monolayer coverage [5].

All absorption measurements were performed using a HP 8453 UV-Visspectrophotometer. Fluorescence measurements on PC films were performedby placing the films on a stationary stage equipped with a fiber-opticmount on a 15 cm-long arm (normal to sample). The output of the fiberwas connected to an Ocean Optics HD2000 spectrofluorometer to measurethe fluorophore emission spectra. The excitation was from the secondharmonic (470 nm) of the diode-pumped Nd:YV04 laser (compact laserpointer design, output power≈30 mW) at angle of 45 degrees. The emissionwas observed through a 500 nm long-pass filter (Edmund Scientific).

The real-co/or photographs of FITC-HSA on non-silvered PC films and PCfilms with SiFs, were taken with a Olympus Digital camera (C-740, 3.2Mega Pixel, 10× Optical Zoom) using the same long-pass filter that wasused for the emission spectra. Time-resolved intensity decays weremeasured using reverse start-stop time-correlated single-photon counting(TCSPC) [14] with a Becker and Hickl Gmbh 630 SPC PC card and anun-amplified MCP-PMT. Vertically polarized excitation at ≈440 nm wasobtained using a pulsed laser diode, 1 MHz repetition rate. Theintensity decays were analyzed in terms of the multi-exponential model:

${I(t)} = {\sum\limits_{i}{\alpha_{i}{\exp \left( {{- t}/\tau_{i}} \right)}}}$

where α_(i) are the amplitudes and τ_(i) are the decay times,Σα_(i)=1.0. The fractional contribution of each component to thesteady-state intensity is given by:

$f_{i} = \frac{\alpha_{i}\tau_{i}}{\sum\limits_{i}{\alpha_{i}\tau_{i}}}$

The mean lifetime of the excited state is given by:

$\overset{\_}{\tau} = {\sum\limits_{i}{f_{i}\tau_{i}}}$

The values of α_(i) and τ_(i) were determined by non-linear leastsquares impulse reconvolution with a goodness-of-fit χ² _(R) criterion.[14]

Initial attempts at directly depositing silver island films (SiFs) ontoplastic substrates resulted in relatively poor silver attachment to thevirgin polycarbonate (PC) surface, FIG. 1—Left. However, after etchingthe PC film in 2 M NaOH for 1 min, followed by silanization providing anamino coating on the surface using APS, 3-(aminopropyl)triethoxysilane,silver island films were readily formed, FIG. 1—right, and could not bewashed from the surface. It is theorized that the strong base providedadditional surface hydroxyl groups for APS attachment by hydrolyzing thePC film, a procedure previously reported by Dauginet, et al [12]. Forthe thin films used in this report, immersion in 2 M hydroxide for 1minute was found to be sufficient for SiF preparation on the plasticsurface, where the SiFs have a maximum optical density around 0.3,consistent with previous reports [3,5]. The plasmon absorption band forthe SiFs was also found to be slightly red-shifted on the PC film ascompared to that typically observed on glass substrates as shown in FIG.2 [3,5].

To test the silver coated plastic surfaces for metal-enhancedfluorescence, unmodified and modified films were equally coated withfluorescein labeled HSA (Human Serum Albumin), as shown in FIG. 3, wherethe FITC-HSA has been shown to be an ideal labeled protein [3,4,5], withregard to positioning the fluorophore a couple of nanometers from thesilver nanoparticles to facilitate metal-enhanced fluorescence [1,2].

FIG. 3 shows that the emission of fluorescein is substantially greateron the silver island film coated modified PC films as compared to anequal coating on PC, but without any SiFs. In addition, no emissioncould be observed from the FITC-HSA coated virgin PC, demonstrating theneed to modify the surface for metal-enhanced fluorescence.Interestingly, even without silver, etching the plastic with hydroxideprovided for a greater protein coverage than the virgin PC film, FIG. 3.The transmission of the cut-off filter used is also shown in FIG. 3 andaccounts for the sharp rising edge of the emission spectra.

The metal-enhanced fluorescence from the silver coated modified plasticfilm was found to be approximately 7 times greater than the modified PCfilm but with no SiFs (i.e. the control sample). This relatively largeincrease in emission intensity could also be seen visually, FIG. 4,through the same long pass filter as used in FIG. 3. As the 470 nm laserexcitation is moved from the unsilvered plastic (Middle) to the silveredplastic side, (Bottom), a dramatic increase in fluorescein emission wasseen.

Metal-enhanced intensity, accompanied fluorescence resulted in bothincreased emission intensity, accompanied by a reduction in fluorophorelifetime, i.e. a radiative modification [1-5]. FIG. 5 shows thereduction in lifetime on the SiFs as

TABLE

Analysis of the Intensity Decay if FITC-HSA on Silvered and UnsilveredModified PC, Measured Using the Reverse Start-Stop Time-CorrelatedSingle Photon Counting Technique and the Multi-Exponential Model τ

τ₂ τ₃ τ (τ) Sample α

(ns) α₂ (ns) α₃ (ns) (ns) (ns) χ_(R) ² FITC- 0.290 0.090 0.330 0.9460.380 3.54 3.00 1.68 1.14 HAS on SiFs FITC- 0.079 0.289 0.289 1.1820.632 3.50 3.16 2.58 0.89 HAS on PC

indicates data missing or illegible when filedplastic. The amplitude weighted lifetime was found to decrease from 2.58ns on the unsilvered plastic to 1.68 ns on the silvered plastic, FIG. 5and Table I.

The photostability of the FITC-HSA was measured on both the unsilveredand silvered modified PC film, FIG. 6. Using the same laser powersignificantly more fluorescence was observed from the silvered plastic,by simply considering the integrated area under the respective curves,FIG. 6—Top. However, when the laser power on the silver surface wasattenuated to give the same initial emission intensity as observed onthe unsilvered but modified plastic, similar photostabilitycharacteristics, FIG. 6—bottom were noticed.

By base hydrolysis of thin polycarbonate films more surfacefunctionality for silver deposition was provided. Subsequently, bycoating these silvered surfaces with a labeled protein, metal-enhancedfluorescence was observed in an approximate 7-fold increase influorescein emission intensity observed from modified and silveredplastic as compared to a modified but unsilvered PC film. Further, bycomparing the emission intensity from the virgin PC film to the modifiedand silvered film, a substantial increase in fluorophore emissionintensity can be realized. Given the widespread use of plasticsubstrates in fluorescence based clinical assays and in drug discovery(e.g. HTS well plates), then simple surface modifications of plasticscould facilitate silver depositions for metal-enhanced fluorescence.Alternatively, unmodified hydrophilic plastics with an abundance ofsurface hydroxyl or even amino groups [15] could be ideal for silverdeposition and alleviate the need for surface plastic modification. Inaddition, surface plastic modification using specific light wavelengthsto break covalent bonds and therefore provide for additional polymersurface functionality will be applicable to modification according tothe present invention. This reagentless approach could readily be usedto prepare plastics for silver deposition.

REFERENCES

The contents of all references cited herein are hereby incorporated byreference herein for all purposes.

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That which is claimed is:
 1. A fluorescence sensing device comprising: amodified polymeric surface comprising amino-terminated functionalgroups; nanometer sized metallic particles positioned on the modifiedpolymeric surface, wherein the metallic particles can be any geometricshape; and a fluorophore compound communicatively linked, via a spacer,to the metal particles at a sufficient distance to enhance thefluorescence of the fluorophore when exposed to electromagneticradiation from an electromagnetic source.
 2. The fluorescence sensingdevice according to claim 1, further comprises a detection device fordetecting fluorescence emissions.
 3. The fluorescence sensing deviceaccording to claim 1, wherein the polymeric surface is polycarbonate. 4.The fluorescence sensing device according to claim 1, wherein theelectromagnetic energy is in the ultraviolet range.
 5. The fluorescencesensing device according to claim 1, wherein the polymeric surface isfabricated of polyamide, polycarbonate, polyester, polyetherimide,polyimide, polynitrocellulose, polyethylene, polypropylene,poly(ethylenevinylacetate), poly-2-pentene, polyphenylene oxide,polyphenylene sulfide, polysulfone, and polystyrene.
 6. The fluorescencesensing device according to claim 1, wherein the spacer is an amino acidor nucleotide sequence.
 7. The fluorescence sensing device according toclaim 1, wherein the modified polymeric surface has been modified with achemical or physical etching agent thereby providing an increased numberof exposed hydroxyl groups or amine group on the polymeric surfacerelative to an unmodified polymeric surface and wherein modifiedpolymeric material is coated with an amine containing silane to providean amino-terminated polymeric material for binding with deposited noblemetals.
 8. The fluorescence sensing device according to claim 7, whereinthe amine-containing silane is selected from the group consisting of3-(aminopropyl)triethoxysilane (APS), dimethylsilyldimethyl amine(DMSDMA), 1,1,3,3-tetramethyl disilazane (TMDS), N,N-dimethylaminotrimethylsilane (TMSDMA), N,N-diethylaminotrimethylsilane (TMSDEA),hexamethyldisilazane (HMDS), Bis(dimethylamino)methylsilane,Bis-(dimethylamino)dimethylsilane and1,1,3,3,5,5-hexamethylcyclotrisilazane (HMCTS).
 9. The fluorescencesensing device according to claim 7, wherein the amine-containing silaneis 3-(aminopropyl)triethoxysilane (APS).
 10. The fluorescence sensingdevice according to claim 7, wherein the chemical etching agent isselected from the group consisting of NaOH, NH₄OH, LiOH, KOH orN(CH₃)₄OH.
 9. The fluorescence sensing device according to claim 7,wherein the chemical etching agent is NaOH and the etching is conductedat a temperature ranging from about 15° C. to about 45° C. for a timeranging from about 1 minute to about 1 hour.
 10. The fluorescencesensing device according to claim 7, wherein the physical etching agentis a dose of UV radiation, in the presence of oxygen.
 11. Thefluorescence sensing device according to claim 1, wherein the device isin the form of a kit comprising a container comprising a layer ofimmobilized metal particles deposited on a modified polymeric substrate,wherein an immobilized probe is connected to the metal particles andwherein the immobilized probe has an affinity for the target molecule;and a fluorophore having an affinity for the target molecule, whereinthe binding of the target molecule to both the immobilized probe andfluorophore causes the fluorophore to be positioned a sufficientdistance from the immobilized metal particles to enhance fluorescenceemission.